Method for inactivating gonadotrophs

ABSTRACT

Certain toxic compounds (T) such as, for example, compounds based upon diphtheria toxin, ricin toxin, pseudomonas exotoxin, α-amanitin, pokeweed antiviral protein (PAP), ribosome inhibiting proteins, especially the ribosome inhibiting proteins of barley, wheat, corn, rye, gelonin and abrin, as well as certain cytotoxic chemicals such as, for example, melphalan and daunomycin can be conjugated to certain analogs of gonadotropin-releasing hormone to form a class of compounds which, when injected into an animal, destroy the gonadotrophs of the animal&#39;s anterior pituitary gland. Hence such compounds may be used to sterilize such animals and/or to treat certain sex hormone related diseases.

RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 07/837,639, filed Feb. 14, 1992, now U.S. Pat. No. 5,378,688entitled "GnRH Analogs for Destroying Gonadotrophs" which is acontinuation-in-part of U.S. patent application Ser. No. 314,653, filedFeb. 23, 1989, now abandoned and also entitled "GnRH Analogs forDestroying Gonadotrophs."

FIELD OF THE INVENTION

The present invention generally relates to methods for sterilizinganimals and to methods for medically treating certain sex hormonerelated diseases such as, for example, cancer of the breast or prostate.More particularly, this invention relates to sterilization and medicaltreatment by means of chemical attack upon the pituitary gland.

BACKGROUND OF THE INVENTION

Considerable interest exists with respect to the subject ofsterilization of animals. This is especially true of those concernedwith veterinary medicine and animal husbandry, particularly as theyrelate to the subject of sterilization of domestic animals such as dogs,cats, cattle, sheep, horses, pigs, and the like. Various methods havebeen developed over the years to accomplish sterilization. For example,with respect to male cattle, the most widely used procedure foreliminating problems of sexual or aggressive behavior is sterilizationthrough surgical castration. This is done in various ways, e.g.,crushing the spermatic cord, retaining the testes in the inguinal ring,or use of a rubber band, placed around the neck of the scrotum, to causesloughing off of the scrotum and testes. However most of these"mechanical" castration methods have proven to be undesirable in onerespect or another; for example they (1) are traumatic, (2) introducethe danger of anesthesia, (3) are apt to produce infection, and (4)require trained personnel. Moreover, all such mechanical castrationmethods result in complete abolition of the testes and this of courseimplies complete removal of the anabolic effects of any steroids whichare produced by the testes and which act as stimuli to growth andprotein deposition.

These drawbacks have caused consideration of various alternativesterilization techniques such as the use of chemical sterilizationagents. However, the use of chemical sterilization agents has its ownset of advantages and disadvantages. On the positive side, chemicalsterilization eliminates the stress and danger associated withmechanical castration. Chemical sterilization also has the addedadvantage of allowing for retention of certain anabolic effectsresulting from a continued presence of low levels of circulatingtestosterone. This is especially valuable in the case of animals raisedfor human consumption since circulating testosterone promotes growth,efficiency of feed conversion and protein deposition. Unfortunately,there are several disadvantages associated with chemical sterilization.For example chemical sterilization is often temporary rather thanpermanent; it also sometimes produces extremely severe, and even fatal,side effects.

Many of these chemical sterilization methods have been aimed atregulation of luteinizing hormone produced at various stages of ananimal's sexual development. For example, with respect to cattle it hasbeen established that in the case of the infantile calf, luteinizinghormone is rarely discharged and testicular production of androgens isat low levels. On the other hand, in a prepubertal calf, or an adultbull, discharges of luteinizing hormone from the anterior pituitaryoccur more frequently and the testes produce considerably larger amountsof testosterone and other steroids. It is thought that these conditionsresult from the following factors: (1) decreases in the concentration ofestradiol receptors in the hypothalamus, (2) concomitant increases inthe concentration of estradiol receptors in the anterior pituitary, and(3) increases the number of gonadotropin-releasing hormone (GnRH)receptors in the anterior pituitary. This increase in GnRH receptors isgenerally regarded as a prerequisite for an animal to pass from theinfantile stage to the prepubertal and mature stages of endocrinedevelopment. Hence, based upon these understandings of thehypothalamic-pituitary-testicular axis, several chemical methods havebeen proposed to modify given animals, e.g., a bull calf, in such a waythat it never enters puberty, but still receives stimuli for growth andprotein deposition through the anabolic effects of steroids produced bymodified testes. In any event, most of the chemicals proposed for suchsterilization purposes are hormones or hormone analogs. For example U.S.Pat. No. 4,444,759 teaches the use of a class of peptides analogous toGnRH (i.e., gonadotropin-releasing hormone, and particularly luteinizinghormone-releasing hormone) are capable of inhibiting release ofgonadotropins by the pituitary gland and thereby inhibiting release ofthe steroidal hormones, estradiol, progesterone and testosterone. Itshould also be noted that the terms "GnRH" (gonadotropin-releasinghormone) and "LHRH" (luteinizing hormone-releasing hormone) aresometimes used interchangeably in the literature. For the purposes ofdescribing the prior art both terms may be employed; however, for thepurposes conveying the teachings of our patent disclosure, applicantsprefer the term GnRH and will use it in describing their compounds.

Be that as it may, some prior art chemical sterilization procedures arespecifically adopted to alter luteinizing hormone secretion before theanimal has attained the age of puberty. This is not surprising since therole of luteinizing hormone in sexual maturation is well known.Luteinizing hormone is a gonadotropic hormone found in the anterior lobeof the pituitary gland and, in male animals, it is known to stimulatethe interstitial cells of the testes to secrete testosterone (seegenerally, The Merck Index, 8th edition, p. 560 (1968), Encyclopedia ofChemical Technology, Vol. 7, pp. 487-488 (1951)).

One approach has been to use certain chemicals to produce antibodies inan animal which exhibit cross-reactivity with the gonadotropins producedby the animal's pituitary gland. It is generally thought that with suchearly antigenic stimulation, formation of antibodies is morecontinuously stimulated by the release of endogenous hormones and thatearly immunization with such luteinizing hormone deters the maturationof the gonads and adnexal glands. This, in turn, is thought to inhibitspermatogenesis at the spermatogonial level. For example, U.S. Pat. No.4,691,006 teaches injection of a compound having an amino acid sequenceof at least 20 units for purposes of eliciting formation of antibodieswhich exhibit cross-reactivity with the gonadotropins produced by theanimal's pituitary. With early antigenic stimulation of this kind, theformation of such antibodies is more continuously stimulated by releaseof endogenous hormones. Early immunization with such luteinizing hormonealso deters the maturation of the gonads and adnexal glands. However,the art has also recognized that early immunization of this kind maytend to make the interstitial tissues fibroblastic. It has also beenfound that such early stimulation of the immunologic system leads todevelopment of a high titered antiserum to luteinizing hormone whichremains at relatively high levels. Nonetheless, periodic boosters ofsuch compounds are often necessary even for adult animals sterilizedbefore puberty in order to maintain high levels of the neutralizingantibodies.

Similarly, luteinizing hormone has been administered to animals afterthey have attained the age of puberty in order to atrophy theirreproductive organs and to cause a decrease in libido (see generally, M.Tallau and K. A. Laurence, Fertility and Sterility, Vol. 22, No. 2,February 1971, pp. 113-118, M. H. Pineda, D. C. Lueker, L. C. Faulknerand M. L. Hopwood, Proceedings of the Society for Experimental Biologyand Medicine, Vol. 125, No. 3, July 1967, pp. 665-668, and S. K. Quadri,L. H. Harbers, and H. G. Spies, Proc. Soc. Exp. Biol. Med., Vol. 123,pp. 809-814 (1966). Such treatments also impair spermatogenesis innoncastrated adult male animals by interruption of the spermatogeniccycle.

Other chemical sterilization agents have been specifically designed foruse on female animals. For example, it is well known that certainantigens will produce an antiserum against a requisite estrogen. This isaccomplished by first making an antigen and then injecting said antigeninto an animal for purposes of antiserum production. The animal is thenbled to recover the antiserum. Any female animal of the same species asthe host animal may then be injected with the antiserum at the propertime prior to ovulation and the injected antiserum will cause temporarysterilization of that animal.

Other methods of chemical sterilization have been based upon directchemical attack upon certain cells of the pituitary itself (as opposedto chemical attacks upon the hormone products of such cells) with a viewtoward permanently destroying such cells. Again, this approach issuggested by the fact that follicle stimulating hormone (FSH) andluteinizing hormone (LH) (sometimes referred to as gonadotropins orgonadotropic hormones) are released by the pituitary gland to regulatefunctioning of the gonads to produce testosterone in the testes andprogesterone and estrogen in the ovaries. They also regulate theproduction and maturation of gametes.

Several chemical agents have been proposed for such purposes. However,it has been found that most chemical agents which are in fact capable ofdestroying the gonadotrophs of an animal's anterior pituitary gland alsotend to produce extremely toxic side effects which can severely weaken,and sometimes kill, the treated animal. Hence, with respect to thegeneral subject of chemical sterilization, it can be said that anychemical capable of producing sterilization without, or with minimal,toxic side effects would be of great value in the fields of animalhusbandry, veterinary medicine and wildlife control.

To date, perhaps the closest concepts and/or compounds to thosedescribed in this patent disclosure are found in a publication by Myers,D. A., Murdock, W. J. and Villemez, C. L., entitled Protein-PeptideConjugation By A Two-Phase Reaction: Biochem. J., 227:1 pg. 343 (1985).This reference teaches a sterilization procedure employing a GnRH analogcomparable to that utilized by applicant in one of his more preferredGnRH/toxin conjugate compounds, namely one based upon a GnRH/diphtheriatoxin conjugate. However, there are some very pronounced differences inthe toxin portions of the respective molecules. These differences residein the fact that different parts or portions of the diphtheria toxin areemployed in the respective resulting compounds. More specifically, theconjugate reported by Myers et al. utilized only the toxin domain of thediphtheria toxin molecule while applicant's diphtheria toxins arecharacterized by their possession of the membrane translocation domainof this toxin as well as the toxic domain. The details and significanceof these molecular differences are important to this patent disclosureand will be discussed at greater length in subsequent parts of thispatent disclosure.

However, before leaving this discussion of the GnRH/diphtheria conjugateaspect of the prior art, it also should be noted that in addition to thearticle by Myers et al. noted above, Myers, on another occasion,published additional information concerning his diphtheria toxin-GnRHanalog conjugate. This was done in his Ph.D. thesis at the University ofWyoming in 1987, entitled: "Hybrid toxins: An approach to cell specifictoxicity." This thesis contains basically the same information as theabove-noted 1985 publication, but--of course--in much greater detail.For example, the thesis includes further information on the biologicalactivity of the Myers conjugate. A second part of this thesis addressesmodifications of Myers' diphtheria toxin in a manner similar to thatdescribed above, but using further information published by Colombattiet al. in the Journal of Biological Chemistry 261:3030 (1986).

Another reference of possible interest in this regard was recentlypublished in the INTERNATIONAL JOURNAL OF PHARMACOLOGY 76: R5-R8 bySingh et al. entitled "Controlled release of LHRH-DT from bioerodiblehydrogel microspheres." Generally speaking, it teaches that a naturalGnRH/diphtheria toxin can be used as a vaccine. In this case the LHRH-DTmolecule induces production of antibodies to GnRH which then serve toinactivate endogenous LHRH in the circulation. Without the endogenousLHRH, there is no stimulation of the anterior pituitary gland to secreteLH and the gonads will cease functioning. However, as the antibodytiters fall, endogenous GnRH will again stimulate the anterior pituitarygland, LH secretion and gonadal function will return. Here again, thoseskilled in this art will appreciate that this is an entirely differentapproach from the "direct chemical attack on the pituitary gland"approach taught in this patent disclosure. That is to say that--unlikeSingh's antibody production approach--applicant's conjugate will notgenerate antibodies to GnRH and no neutralization of endogenous GnRHwill occur. Instead, with applicant's approach, the cells in theanterior pituitary gland which are activated by GnRH will be destroyedby direct chemical attack thereon. Moreover, this attack results inpermanent, rather than temporary sterility.

However, before going on to these details, it also should be noted thatknowledge of the above noted sex hormone functions has produced severaladvances in the field of human medicine as well. For example, thepotential for achieving chemical castration (rather than "surgical"castration) with certain luteinizing hormone-releasing hormone (LHRH)analogs has been reported (see for example, Javadpour, N., LuteinizingHormone-Releasing Hormone (LHRH) in Disseminated Prostatic Cancer; 1M,Vol. 9, No. 11, November 1988). Table I below gives the structure ofLHRH and the structure of certain analogs (e.g., Goserelin, Leuprolide,Buserelin and Nafarelin) of LHRH which are capable of temporarilysuppressing luteinizing hormone secretion and thereby suppressing thegonads. As a consequence, these LHRH analogs have come to be regarded asa promising new class of agents for the treatment of varioushost-dependent diseases, especially prostatic cancer. In referring toTable I, it first should be noted that LHRH has a decapeptide structureand that substitution of certain amino acids in the sixth and tenthpositions of the LHRH produce analogs which render agonists that are upto 100 times more potent than the parent LHRH compound (hence thesecompounds are often referred to as "superagonists"). The structures ofLHRH and the most commonly known LHRH superagonists are listed below.

    ______________________________________                                        STRUCTURES OF LHRH AND SOME SUPERAGONISTS                                     (Superagonists have substitutions at                                          positions 6 and 10)                                                            ##STR1##                                                                     SUPERAGONISTS:                                                                Name     Subs. at 6    Subs. at 10                                                                              Terminator                                  ______________________________________                                        Goserelin:                                                                             D-Ser(tBu)    AzaGly     Amide                                       Leuprolide:                                                                            D-Leu         des-Gly    Ethylamide                                  Buserelin:                                                                             D-Ser(tBu)    des-Gly    Ethylamide                                  Nafarelin:                                                                             D-2-NaphthylAla                                                                             None       Amide                                       ______________________________________                                    

While these compounds represent the most promising means for palliativetherapy because of their relative lack of side effects, they areparticularly expensive and must be administered repeatedly. Even thenewest formulations utilizing polymer encapsulated drug or other depotforms will require at least monthly administration. Improved depot formsalso are presently in development, but they too are likely to be equallyexpensive and they too will probably require monthly administration. Inresponse to these many drawbacks, applicants have developed a class ofcompounds which is capable of producing safe, inexpensive, chemicalcastration as an alternative to surgical castration. Such drugs alsogreatly simplify therapy of the generally elderly patients with prostatecancer, and could eliminate the need for surgical castration (stillpreferred by many urologists) as well as provide a medical alternativeto oophorectomy in females with advanced breast cancer. Moreover, as amodel system, the ability to eliminate pituitary gonadotrophs in vivo,which are regulated by GnRH receptors in response to ligand stimulationin a predictable fashion, is a highly appealing first step toward themore complex use of toxins conjugated to antibodies to eliminate tumortargets. Hence, use of applicants' compounds generally will fall intotwo major areas of use. The first is sterilization of mammals of alltypes; the second is chemical castration of mammals in general, andhuman beings in particular, for purposes of treating breast or prostatecancer by ablating those pituitary cells, namely gonadotrophs,responsible for LH secretion.

SUMMARY OF THE INVENTION

The present invention provides a group of GnRH/toxin conjugate compoundsand processes for using them to sterilize mammals (animals and humans)and/or for treating certain sex hormone related diseases such as cancerof the prostate or cancer of the breast. The active parts of thesecompounds or agents may be referred to as "toxic compounds", ("T") or"toxins" for the purposes of this patent disclosure without changing theintended scope of the herein described compounds and/or processes. Inany event, the most effective, and hence most preferred, of these toxincompounds will include: diphtheria toxin, ricin toxin, abrin toxin,pseudomonas exotoxin, shiga toxin, α-amanitin, pokeweed antiviralprotein (PAP), ribosome inhibiting proteins (RIP), especially theribosome inhibiting proteins of barley, wheat, flax, corn, rye, gelonin,abrin, modeccin and certain cytotoxic chemicals such as, for example,melphalan, methotrexate, nitrogen mustard, doxorubicin and daunomycin.All of these toxins are characterized by their inability, in their ownright, to chemically attack the gonadotropin-secreting cells of theanterior pituitary gland as well as by their concomitant ability tochemically attack gonadotropin-secreting cells when conjugated with GnRHmolecules (and GnRH analogue molecules) according to the teachings ofthis patent disclosure.

Some of these toxins (e.g., bacterial toxins and certain plant toxins)can be characterized by whether or not a "whole" molecule of a giventoxin is employed. For the purposes of this patent disclosure the term"whole" may be taken to mean that the molecule has at least a toxicdomain, a translocational domain and a cell binding domain. If, however,one or more of these domains are removed from a "whole" toxin molecule,then the resulting molecule will be characterized as a "modified" toxinor "modified" molecule of that toxin. TABLE I below gives somerepresentative "whole" and "modified" toxins. Some of these toxin types(e.g., bacterial and plant toxins) also can be further characterized bytheir possession of so-called "A-chain" and "B-chain" groups in theirmolecular structures. It also should be noted that the toxic domain isoften referred to as the "A-chain" portion of the toxin molecule whilethe toxic domain, translocation domain and cell-binding domain are oftencollectively referred to as the "whole" toxin or the A-chain plus theB-chain molecules. For example, such further classifications could bemade according to the attributes, categories and molecular sizes notedin TABLE I below (wherein the letters A and B represent the presence ofA-chains or B-chains and the letter K designates the symbol("kilodalton" used to designate molecular sizes of such molecules):

TABLE I Single Chain Toxins

Pokeweed antiviral protein

Gelonin ribosome-inhibiting protein (RIP)

Wheat RIP

Barley RIP

Corn RIP

Rye RIP

Flax RIP

Bacterial Toxins

Diphtheria toxin (whole) having a toxic domain, a translocation domainand a cell-binding domain=62K

Diphtheria toxin (modified) having a toxic domain and a translocationdomain=45K

Pseudomonas exotoxin (whole) having a toxic domain, a translocationdomain and a cell-binding domain=66K

Pseudomonas exotoxin (modified) having a toxic domain and atranslocation domain=40K

Shiga toxin (whole) having a toxic domain, a translocation domain and acell binding domain=68K

Shiga toxin (modified) having a toxic domain=30K

Plant Toxins

Ricin A+B (whole)=62K

Ricin A=30K

Abrin A+B=62K

Abrin A=30K

Modeccin A+B=56K

Modeccin A=26K

Small Chemical Toxins

Melphalan

Methotrexate

Nitrogen Mustard

Daunomycin

Doxorubicin

Applicants have also found that of all the possible toxin moleculesnoted above, the bacterial and plant toxins having both a toxic domainand a translocation domain (which may also be referred to as B-chain"parts", "shortened B-chain, amino acid sequences", etc.), but not acell-binding domain are the most effective--and hence the mostpreferred--conjugate compounds for applicant's sterilization purposes.The procedures by which cell-binding domains can be deleted are ofcourse well known to this art and need not be discussed in any greatdetail.

Moreover, in considering the general subject of transmembrane transportproteins, as they relate to this invention, applicants would also pointout that there are a number of viral proteins, for example, whichfunction in ways similar to the "translocation domain" functions ofdiphtheria toxin, ricin, and of Pseudomonas toxin. These include theSendai virus HN and F glycoproteins, and the Adenovirus penton proteinsalong with similar fusogenic proteins of Semliki Forest virus. Also,lipophilic polylysines, such as poly(1-lysine) conjugated toglutarylphosphatidylethanolamine can function in this way. Consequently,those skilled in the art will appreciate that the transmembranetransport of applicants' conjugates can be enhanced by inclusion of anysuch fusogenic moieties into our GnRH-toxin conjugates.

However, regardless of such concerns for the presence, identity, and/orsize of B-chains in certain toxin molecules, applicants have found thatall of the herein described sterilization agents can be most effectivelydelivered to the pituitary gland if they are chemically conjugated withvarious peptide hormone molecules such as certain analogs ofgonadotropin-releasing hormone, GnRH. Again, this conjugation isnecessary because, for the most part, the above toxins, by themselves,are not capable of binding with cell membranes in general. That is tosay that applicants have found that it is only when a GnRH analog of thetype described herein is linked to a toxin of the types noted above doesthat toxin become capable of binding to cell membranes, and then only tothose cells whose membranes contain receptors for GnRH (i.e.,gonadotrophs in the anterior pituitary gland). Other less preferred, butstill operative peptide hormone molecules (other than applicant'spreferred gonadotropin-releasing hormone analogues) to which the hereindisclosed toxins could be so conjugated for applicant's sterilizationpurposes include: human chorionic gonadotropin, equine chorionicgonadotropin, luteinizing hormone and follicle-stimulating hormone.

At this point, it should again be emphasized that for the purposes ofthis patent disclosure, the term gonadotropin-releasing hormone willusually be abbreviated as "GnRH" and that, for the most part, certainhereinafter described analogs of GnRH are generally more effectivecarrier peptide hormone molecules for the practice of this inventionthan the fundamental or parent GnRH molecule. In their most generalizedsense, these analogs will be abbreviated as "GnRH-A", with the "A"designating that the resulting compound is an analog, "A" of thefundamental GnRH molecule. Again, any general toxin compound which isconjugated with a GnRH-A molecule will be abbreviated by the letter "T"for toxin. Thus, the abbreviation for a generalized conjugate of aGnRH-A analog and a toxin will be "GnRH-A-T".

In the case of GnRH-A carrier peptide molecules, the linking or couplingof the GnRH-A molecule and the T molecule is preferably carried out atthe 6 position of the GnRH-A molecule. This modification may include useof a linkage using a heterobifunctional reagent "Y" which will bedescribed in much more detail in subsequent portions of this patentdisclosure. That is to say that the most preferable technique forproduction of the resulting GnRH-A-T conjugate molecule will involvemodification of the 6 position of the fundamental GnRH molecule. Inother words, amino acid substitutions at the 6 position of thefundamental GnRH molecule will yield analogs with particularly highaffinities for GnRH receptors on cells of the pituitary gland andthereby providing an improved means for introducing the toxin into thetargeted cells.

The most preferred amino acids for substitution at the 6-position willinclude lysine, D-lysine, aspartic acid, D-aspartic acid, glutamic acid,D-glutamic acid, cysteine, D-cysteine, ornithine, D-ornithine, tyrosine,D-tyrosine as well as other amino acids having suitable side-Chainfunctional groups such as, for example, amino groups, carboxylic groups,hydroxyl groups or sulfhydryl groups. Similarly the 10 position of thefundamental GnRH molecule can be modified to produce other analogvariations useful for applicant's purposes. The substituents mostpreferred for this purpose will include Gly-NH₂, ethylamide andAzA-Gly-NH₂.

Heterobifunctional reagent Y is, most preferably, used to link a GnRH-Agroup or moiety to a toxic group or moiety T. Most preferably such toxicgroups T and their associated GnRH-A carrier peptide molecules will becovalently linked by a linking or coupling agent selected from the groupconsisting of 2-iminothiolane, N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP),4-succinimidyloxycarbonyl-α-(2-pyridyldithio)-toluene (SMPT),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-diazobenzidineand glutaraldehyde.

Given all of these structural concerns, a generalized chemicalstructural diagram of an amino acid sequence of a GnRH molecule and of agroup of highly preferred resulting GnRH-A-T carrier peptide moleculesfor the practice of this invention could be depicted as follows:##STR2## wherein X is an amino acid, Y is a linking group, Z is achemical substituent selected from the group consisting of Gly-NH₂,ethylamide and Aza-Gly-NH₂ and T is a toxin group selected from thegroup consisting of the plant toxins: ricin, modeccin, abrin, pokeweedanti-viral protein, α-amanitin, gelonin ribosome inhibiting protein("RIP") barley RIP, wheat RIP, corn RIP, rye RIP and flax RIP; thebacterial toxins selected from the group consisting of: of diphtheriatoxin, pseudomonas exotoxin and shiga toxin (and especially thosebacterial toxins having a toxic domain and a translocation domain) andthe chemical toxins selected from the group consisting of: melphalan,methotrexate, nitrogen mustard, doxorubicin and daunomycin.

Those skilled in this art will appreciate that some specific compoundsfalling within the above generalized structure are often referred to as"D-Lys⁶ -GnRH." That is, in normal peptide nomenclature, the referenceto D-Lys⁶ before the GnPH indicates that the normal 6-position aminoacid group of the GnRH molecule (i.e., a "Gly" group), has been replacedby lysine. Thus, the X, i.e., the 6-position X amino acid would in factbe lysine. Hence, the most general GnRH-A amino acid sequence could bedepicted as follows: ##STR3##

That is to say that, applicant's molecules will be further characterizedby having a generalized amino acid in the X (or 6) position. Preferably,this amino acid will be selected from the group consisting of: lysine,D-lysine, ornithine, D-ornithine, glutamic acid, D-glutamic acid,aspartic acid, D-aspartic acid, cysteine, D-cysteine, tyrosine andD-tyrosine.

Within the possibilities implicit in the general structure, aparticularly preferred GnRH analog would be:

(Eq. III)

    pyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-ethylamide

This molecule also could be referred to as [D-Lys⁶ -des-Gly¹⁰]-GnRH-ethylamide and, regardless of nomenclature, it represents one ofapplicant's most preferred GnRH-A molecules.

The presence of the Y component of the most general structure (i.e.,Equation I) is optional--but highly preferred. Again, if used, such Ygroups are most preferably selected from the group consisting of:2-iminothiolane, N-succinimidyl-3-(2-pyridyldithio) proprionate (SPDP),4-succinimidyloxycarbonyl-α-(2-pyridyldithio)-toluene (SMPT),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodimide (EDC), bis-diazobenzidineand glutaraldehyde.

The most preferred forms of these compounds will have an amino group, acarboxylic group and/or a sulfhydryl group, to aid in the Y group'sperformance of this GnRH-A to T linking function. In other words the Tgroup most preferably will be attached to a GnRH-A molecule by means ofan amino, carboxylic or sulfhydral group of 2-iminothiolane,N-succinimidyl-3-(2-pyridyldithio) proprionate (SPDP),4-succinimidyloxycarbonyl-α-(2-pyridyldithio)-toluene (SMPT),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-diazobenzidineand glutaraldehyde. Similarly, the Y group most preferably will beattached to the X group at the site of an amino group, a carboxylicgroup, a sulfhydryl group or a hydroxyl group of whatever amino acidgroup is employed at the 6-position of applicant's GnRH-A molecule.

As previously noted, the T group represents a toxin group which, firstand foremost, is capable of chemically attacking the gonadotrophs of thepituitary gland when conjugated to the carrier peptide (GnRH-A)molecules described in this patent disclosure. Again, as seen in TABLEI, certain toxins T such as the bacterial toxins and plant toxins suchas ricin, abrin and modeccin, can be composed of a toxic domain (alsoreferred to as an A-chain), a translocation domain and a cell-bindingdomain (the latter two domains are sometimes referred to as the B-chain)and that applicants believe that, in general, use of toxins having atoxic domain plus a translocation domain, but not a cell-binding domain,will give more effective results than use of toxins having only a toxicdomain (A-chain) only or a toxic domain, translocation domain andcell-binding domain (also referred to as whole toxin or A-chain plusB-chain). The ribosome-inhibiting proteins (RIP) also will be effectivetoxins, but here again, only after conjugating them to a GnRH analog.That is to say that by themselves, they are not toxic since they do notcontain a cell membrane binding domain. However, if conjugated to one ofapplicant's GnRH analogs, the resulting conjugate molecule can interactwith GnRH receptors and gain entry into the pituitary cell, therebypreventing protein synthesis and ultimately causing the desiredeffect--cell death. The RIPs of barley, corn, wheat, rye and flax willbe especially useful for this purpose. Pokeweed antiviral protein issimilar in nature to the RIPs noted above and hence can also be employedas the toxin T. The bacterial toxins, diphtheria toxin, pseudomonasexotoxin and shiga toxin are especially preferred. Again, thesebacterial toxins are originally comprised of a toxic domain, atranslocation domain and a cell-binding domain, but applicants havefound that in general those having their toxic domain plus theirtranslocation domain are generally more effective than those bacterialtoxin having only a toxic domain or those comprised of the wholemolecule. Chemical toxins selected from the group consisting ofmelphalan, methotrexate, nitrogen mustard, doxorubicin and daunomycinare particularly preferred. Obviously, A-chain and B-chainconsiderations will not be applicable to "chemical" toxins because theyare not made up of amino acid groups such as those found in bacterial orplant toxins.

It should, however, also be noted that regardless of whether the toxin Tis comprised of an A-chain, an A-chain plus a portion of a B-chain, or achemical molecule which does not contain an amino acid sequence, it ispreferably attached to the GnRH portion of the overall conjugatemolecule via a linking Y compound selected from the group consisting of:2-iminothiolane, N-succinimidyl-3-(2-pyridyldithio) proprionate (SPDP),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT),m-maleimidoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-diazobenzidineand/or glutaraldehyde.

It should again be emphasized that one particularly important aspect ofthe herein disclosed invention is based upon applicant's finding thatthose appropriate (i.e., bacterial or plant) toxin moieties having bothan A-chain and at least a portion of a B-chain, but not all of theB-chain, in the overall GnRH/toxin conjugate molecules are especiallywell suited to the herein described sterilization functions. Thispreference for the presence of a portion of a given toxin's B-chain inthe overall conjugate molecule is important to this patent disclosurefor several reasons. First, applicant's B-chain-containing compoundshave proven to be generally much more effective sterilization agentsthan those amino acid containing toxins having only an "A-chain"portion. Moreover, such amino acid containing toxins also tend to beless toxic in their side effects.

This difference also serves to distinguish applicant's invention fromthose other sterilization methods using GnRH molecules in their ownright or from those employing other GnRH/toxin conjugate compounds. Forexample, the previously noted GnRH/diphtheria toxin used in the processreported by Myers et al. utilized only the A-chain portion of thediphtheria toxin molecule. That is to say that diphtheria toxin is a 62kilodalton protein, composed of a 21 kilodalton A chain and a 37kilodalton B chain linked together by disulfide bonds. Myers et al, ineffect, confirmed that an A-chain, diphtheria toxin can serve to inhibitprotein synthesis in a cell by catalyzing the ADP-ribosylation of a cellconstituent known as "elongation factor 2." Again, in the absence ofprotein synthesis, a cell cannot function and eventually dies.

This follows from the fact that a cell's elongation factor 2 is locatedin its cytoplasm, and a toxin such as diphtheria toxin must first gainentry into the cytoplasm in order for its toxicity to be manifested.Thus, the most preferred forms of toxins for the practice of thisinvention (e.g., use of diphtheria toxin in applicant's resultingGnRH-A-T conjugates) will have a toxin molecule which includes the toxicdomain (for cytotoxicity) and the translocation domain that increasesthe ability of the overall molecule to cross cell membranes. That is tosay that this translocation domain "portion" serves to greatly assistsentry of the toxic domain portion of the toxin into a cell's cytoplasmand thus increases the potency of the resulting conjugate as asterilization agent.

Applicant has, however, found that the presence of the translocationdomain of a toxin such as diphtheria toxin greatly enhances thesterilization efficacy and/or nontoxicity of GnRH-A-T conjugates of thetype disclosed in this patent application. Again, use of an entire toxinmolecule is not preferred for applicant's purposes. That is to say thatin those cases where an overall toxin molecule contains a toxic domain,a translocation domain and a cell-binding domain, applicant prefers todelete the cell-binding domain.

For example, a diphtheria B chain has two parts, a translocation domainand a cell-binding domain. These two portions are a carboxyl terminal of8 kilodaltons which contains a cell surface binding domain that permitsdiphtheria toxin to attach to nearly all mammalian cells to which it isexposed and an amino terminal of 21 kilodaltons which contains severalhydrophobic regions that can insert into a membrane at a low pH. Thecell-binding domain of the diphtheria's B-chain is preferably cleavedaway.

As previously noted, in some of the most preferred conjugate molecules,applicant has provided a diphtheria toxin portion comprised of a toxicdomain and a translocation domain and additionally comprising a "spacer"group which most preferably ends in a cysteine residue. This arrangementhas the advantage of providing a free sulfhydryl group that can be usedto attach the toxin molecule to the GnRH analog in such a way as tominimize interference with the desired enzymatic activity (i.e.,performance of the toxicity function of the toxic domain).

Again, applicant has discovered that the analogue of the GnRH moleculehaving the following structure:

    pyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-ethylamide

is particularly efficacious for conjugation and delivery of a diphtheriatoxin comprised of an A-chain and a part or fragment of the diphtheriatoxin molecule's B-chain amino acid sequence. As previously noted, thismolecule could be referred to as the [D-Lys⁶ -des-Gly¹⁰]-GnRH-ethylamide analogue of the GnRH molecule. Regardless ofnomenclature, applicant has found this to be the most effective (and,hence, the most preferred) GnRH analogue/diphtheria toxin conjugate forapplicant's sterilization methods. And, as in the more general casesnoted in the previous discussion of the nature of the 6 position "X"group of the more general molecular structures, lysine, D-lysine,ornithine, D-ornithine, glutamic acid, D-glutamic acid, aspartic acid,D-aspartic acid, cysteine, D-cysteine, tyrosine and D-tyrosine couldeach be substituted in the amino acid #6 position of this most preferred[D-Lys⁶ -des-Gly¹⁰ ]-GnRH ethylamide/diphtheria molecule. However, italso should be noted that the analogs resulting from these changes atthe 6 position are generally somewhat less preferred, but still useful,for applicant's general process.

The resulting conjugates are specifically targeted to thegonadotropin-secreting cells of the anterior pituitary gland. Indeedthey are the only cells to which the gonadotropin-releasing hormoneportion of applicant's conjugates will bind. Hence, the toxic compounds,bound to an analog of gonadotropin-releasing hormone, serve topermanently destroy a subpopulation of the anterior pituitary cells andthereby eliminate the gland's ability to secrete gonadotropins.Applicant has termed this mechanism "direct chemical attack" to contrastit with the use of certain GnRH molecules to elicit an immune responseto the gonadotropin products of the pituitary. This direct chemicalattack upon the pituitary gland, in turn, causes the animal's gonads toatrophy and lose their ability to function for reproductive purposes. Inother words, without functioning gonadotrophs, an animal is not able tosecrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH)and thus is rendered sterile. Applicants have postulated that thecompounds of this patent disclosure inhibit synthesis of LH, andpresumably other proteins made by gonadotrophs, because they tend toinhibit all protein synthesis once these compounds gain entry into thepituitary cells.

Consequently, these compounds have great potential utility in humanmedicine as well as in veterinary medicine. This follows from the factthat there are several important biological reasons for employingcastration and antifertility drugs in humans. For example, breast andprostate cancers are but two examples of sex steroid-dependent tumorswhich respond to such hormonal manipulation. At present, the onlyreliable way to inhibit steroid-dependent tumor growth is throughadministration of counter-regulatory hormones (e.g., DES in prostatecancer), sex-steroid hormone binding inhibitors (e.g., tamoxifen inbreast cancer) or surgical castration. Thus the potential medical usesof such chemical castration compounds are vast and varied. For example,prostate cancer remains an important cause of cancer deaths andrepresents the second leading cancer of males. The present palliativetreatment for advanced prostate cancer cases involves reduction of serumtestosterone/DHT levels through use of surgical castration. It shouldalso be noted that for purposes of disease and/or fertility control,especially in humans, it may be desirable to use applicants' compoundsto ablate pituitary gonadotrophs in conjunction with other modes oftreatment. For example, it is anticipated that chronic administration ofprogestins and estrogens to females and androgens to males might benecessary to prevent loss of secondary sex characteristics, behavior andosteoporosis. However, through judicious use of the herein disclosedcompounds, especially in combination with appropriately administered sexsteroids, desirable antifertility effects can be achieved. Another areaof application in human medicine is treatment of endometriosis. Thiscondition, which produces painful growth of endometrial tissue in thefemale peritoneum and pelvis also responds to inhibition of sex steroidsynthesis. Those skilled in this art will also appreciate that theherein disclosed compounds could be used to partially reduce sex-steroidsecretions, and thus reduce or eliminate certain hormone relatedbehavior problems while retaining improved growth stimulation.

The dose/time adjustments associated with the use of these compounds canvary considerably; however, these compounds are preferably administeredby injection into a mammal in concentrations of from about 0.1 to about10 milligrams per kilogram of the mammal's body weight. Sterilizationmay be accomplished with as few as one injection; but multipletreatments (e.g., based upon concentrations of from about 0.03milligrams once every 4 days to about 1 milligram per kilogram of bodyweight for 20 days) are alternative sterilization schemes. Furthermore,as sterilization agents, the compounds of this patent disclosure can beused before or after puberty. They too are especially useful in thoseareas of animal husbandry where the anabolic benefits of non-surgicalsterilization techniques can contribute to meat production and/orquality. In one preferred embodiment of this invention the compounds ofthis invention are administered to male cattle between the ages of about8 weeks and 20 weeks at least once and in a concentration of from about0.1 to about 10 milligrams per kilogram of the animal's body weight.

The toxic moieties T of the herein disclosed compounds are obtainablefrom both natural and synthetic sources. For example, pokeweed antiviralprotein can be isolated from leaves of pokeweed plants and purified bygel filtration chromatography. It can then be, by way of example,conjugated to D-Lys⁶ -desGly¹⁰ ]-GnRH-ethylamide via the amino group onthe lysine and through a sulfhydryl group introduced into the pokeweedantiviral protein by a heterobifunctional reagent. In any event, one ofthe chief advantages of these compounds is their ability to producepermanent sterilization without strong toxic side effects. Hence thesecompounds may be used on mammals such as human beings, domestic animals,pets or wild animals. Moreover, they can be administered as a singleinjection which can induce permanent and irreversible sterility in bothmale and female mammals. However, an alternative approach to achievesterilization is through multiple injections at lower dosages than thoseemployed in a single treatment or by slow release implants (i.e.,biodegradable formulations).

Applicants also have postulated that the "B-chain" portion of theirtoxic moieties are important not only for binding to cell surfaces, butfor trans-membrane translocation of their A-chain. This was particularlydemonstrated for the A-chain of Diphtheria toxin, Ricin and Pseudomonasexotoxin. To this end, applicants prepared conjugates of GnRH-A to A andB chains of Diphtheria toxin as well as to a modified A-B chain whichwas genetically engineered to eliminate the carboxy terminal bindingportion of the B-chain. These conjugates were shown to bind to pituitarycell GnRH receptors. They also were found to possess enhanced toxicityover A-chain conjugates based on improved trans-membrane transportcharacteristics. Given this, those skilled in the art will appreciatethat numerous genetic and chemical modifications of B-chains shouldallow further exploitation of this approach. That is to say that, bysuch methods, it is possible to generate a whole series of conjugatesthat can be characterized as GnRH-A-A/B, GnRH-A-A, GnRH-A-A plus GnRH-B,all of which could enhance the findings described herein by simultaneousdelivery of membrane active B-chains with the herein described GnRH-A-Tconjugates.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B respectively depict the results of GnRH inducedsecretion of LH based upon a single injection of a GnRH-A-T compound andthe results of GnRH induced secretion of LH based upon 4 injections of aGnRH-A-T compound.

FIGS. 2A and 2B indicate inactivation of certain grain hemitoxins (wheathemitoxin and barley hemitoxin) by SPDP conjugation.

FIG. 3 depicts the results of a SDS-PAGE analysis of carbodiimideconjugated hemitoxins.

FIG. 4 shows the inhibition of 2-iminothiolane-conjugated barleyhemitoxin.

FIG. 4A shows SDS-PAGE analysis of barley hemitoxin after conjugation to[D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide using 2-iminothiolane.

FIG. 5 shows binding curves indicating the ability of [D-Lys⁶, des-Gly¹⁰]-GnRH-ethylamide toxin conjugates to bind to pituitary receptors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

One of the chief objects of this invention is to provide a class ofcompounds which will allow safe, inexpensive, chemical castration. Assuch, applicants' compounds represent an alternative to surgicalcastration as well as to surgery for treatment of diseases such asbreast cancer or certain sex hormone related prostate cancers. In orderto better define this class of compounds, Applicants conducted studieson various linking technologies as they apply to numerous toxincandidates. These studies resulted in the herein disclosed group ofconjugate compounds. In general these compounds display good gonadotrophmembrane binding characteristics along with retention of toxin activity.

In general, the sterilization activity of the compounds of this patentdisclosure was tested in receptor binding assays (to be sure a givenconjugate was still capable of interacting with the GnRH receptor cellsof the pituitary), in a cell-free translation system (to insure that thetoxic protein maintained its toxicity), in cell culture systems (todetermine if a given toxic conjugate is capable of inhibiting synthesisof LH), and in test animals (to determine if sterility was induced). Forexample, one of the more effective of these sterilization agents was a[D-Lys⁶ -des-Gly¹⁰ ]-GnRH-ethylamide which was conjugated to pokeweedantiviral protein using carbodiimide as the "linkage" group Y betweenthe carrier protein molecule and the toxin moiety.

Again, a distinct advantage of each of the sterilization agents of thisinvention, and pokeweed antiviral protein in particular, is that theyhave an extremely limited ability to enter cells in an animal's bodyunless they are first conjugated to a carrier such asgonadotropin-releasing hormone. Such conjugation was accomplished inseveral ways. By way of example, pokeweed antiviral protein can beconjugated to a [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide molecule via theε-amino group on the D-lysine to a sulfhydryl group on the pokeweedantiviral protein.

By way of further information, applicants found that this type oflinkage reduces the ability of the [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamideto bind to the GnRH receptor by 99%. In addition, the conjugationprocedure reduces the toxicity of the pokeweed antiviral protein by 99.5in a cell-free translation system. However, despite large reductions inactivity of both the GnRH analog and the sterilization agent by thisparticular conjugation procedure, some activity of each was maintained.The activity of this conjugate was also tested in a pituitary cellculture system. In this system, pituitary cells were incubated with thesterilization agent conjugated to [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamidefor 16 hours. After incubation, the sterilization agents were removedfrom the incubation media by extensive washing and the cells were thencultured for an additional 24 hours. The increase in total LH, i.e.,that present in the media plus that in the cells during the 24 hourperiod, represents the ability of the treated cells to synthesize LH.Using this system, it was established that these toxic conjugates cancompletely inhibit synthesis of LH by the cultured cells. Thus, by thismethod, it was established that the compounds of this patent disclosurecan inhibit synthesis of LH and presumably other proteins made bygonadotrophs since this class of compounds has the ability to inhibitall protein synthesis once they gain entry into a cell.

Applicants also tested these compounds using an in vivo model. The testsystem initially chosen was the ovariectomized female rat. The parameterexamined was GnRH induced secretion of LH. The results of such anexperiment with rats are shown in FIG. 1A. It indicates that a singleinjection of a toxic conjugate (i.e., GnRH-A-T) wherein the toxic moiety(T) pokeweed antiviral protein and the GnRH-A moiety was [D-Lys⁶,des-Gly¹⁰ ]-GnRH-ethylamide. During week 1, this compound inducedsecretion of LH equivalent to that of GnRH-A alone. This indicated thatthe sterilization agent conjugate was binding to the GnRH receptor invivo. During week 2, release of LH was reduced by 50% in the GnRH-Atreated group (controls), but by >90% in the GnRH-A-T group. By thethird week, the release of LH in the GnRH-A-T group had returned to thesame level as that observed in the control animals. This indicated thata single treatment with the sterilization agent conjugate was probablynot sufficient to completely kill the gonadotrophs in vivo. It mighthowever be the basis for a temporary sterilization. Based upon thisfinding, a second experiment was conducted to examine the effect of 4injections of a pokeweed antiviral sterilization conjugate at 3-dayintervals on the ability of ovariectomized rats to release LH. In thisexperiment, the rats were unable to release LH in response to GnRHstimulation one month after initiation of the treatment (FIG. 1B). Thesedata strongly indicate the ability of these conjugates to permanentlyinhibit reproduction in intact male and female animals.

In another set of experiments, intact rats were given 4 injections ofGnRH-A-T compounds, again wherein the toxic moiety T was selected frompokeweed antiviral protein, ricin A chain, and ribosome inhibitingproteins, of certain grains (again, those of wheat, corn, barley andrye,) at 3-day intervals and their subsequent reproductive capacity wascompared to rats treated with only the respective toxin T or to that ofuntreated rats. In this experiment, treatment of male rats with only thetoxin T did not reduce their fertility compared to controls (percentageof females that became pregnant was 100%). However, fertility wasgreatly reduced in those males that were treated with a GnRH-A-T agentsuch as, for example [D-Lys⁶ -des-Gly¹⁰ ]-GnRH-ethylamide conjugated topokeweed antiviral protein, i.e., only 50% of the females exposed tomales became pregnant. Moreover, fertility did not appear to increasewith time after treatment. Histological examination of the testes ofthese rats indicated that most of the seminiferous tubules were devoidof sperm. However, 10% of the tubules appeared to still be producingsperm and probably accounted for the pregnancies observed. The weight ofthe testes was reduced by nearly 50% and did not recover within 6 monthsafter the end of treatment. Thus, the effects of the treatment appearedto be permanent and dose related. Female rats treated with the toxicconjugate were sterile and remained so for at least 4 months (i.e.,about 30 reproductive cycles) after the end of treatment. Most importantis the fact that none of the rats treated with the toxic conjugateappeared to have any side effects.

Exemplary Chemical Experimental Methods

1. Synthesis of [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide. Synthesis of thisanalogue was accomplished using the solid phase method on hydroxymethylresin and cleavage from the resin by ethyl amine, yielding theethylamide. Following HF cleavage of protecting groups from side chainsthe peptide was purified by countercurrent distribution, purity of thepeptide was assured by TLC, paper electrophoresis, and amino acidanalysis of the acid hydrolysate.

2. Applicants also produced a caproic acid derivative (134.91 mg) andthe lysosomal hydrolase sensitive tetrapeptide spacer Leu-Ala-Leu-Ala-DLys⁶ (16.25 mg).

3. Conjugation of [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide to toxins usingSPDP. Applicants endeavored to construct toxic conjugates of [D-Lys⁶,des-Gly¹⁰ ]-GnRH-ethylamide with the ricin A-chain. At the time thesestudies were initiated, Ricin A-chain was commercially available, butapplicants found it to be both expensive and very unstable totemperature changes or conjugation procedures. Construction of aneffective hemitoxin [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide conjugaterequires coupling of hemitoxin to hormone via a protein cross-linkingreagent that does not block either the enzymetic activity of thehemitoxin or the binding specificity of the hormone. Therefore,applicants investigated a number of different hemitoxins in addition toricin A and pokeweed antiviral protein and a number of differentconjugation techniques. This work was largely directed at purificationof certain plant hemitoxins, i.e., ribosomal inhibitory proteins,("RIP"), a relatively recently recognized group of proteins which sharethe ability to enzymically inactivate mammalian ribosomes. Such toxinsare potentially promising as alternatives to the more familiar A-chainsof, for example, ricin in that they do not require separation from thecell-binding B-chains. The bi-functional coupling reagent most commonlyused for this purpose is N-succinimidyl 3-(2-pyridyldithio) propionate(SPDP). This compound forms covalent linkages to either free amino orsulfhydryl groups on proteins, but SPDP normally is attached to aminogroups in hemitoxins, partly because many hemitoxins do not containsylfhydryls that are available for coupling.

Initial experiments examined the reaction of SPDP with both the wheatand barley hemitoxins at various SPDP: hemitoxin ratios. The reactionswere carried out at pH 9 for 30 minutes at 23° C. at a proteinconcentration of 0.6 mg/ml. After 30 minutes a 20-fold molar excess(over SPDP) of lysine was added to react with free SPDP and thehemitoxins diluted and assayed for inhibition of polyphenylalaninesynthesis on Ehrlich ascites cell ribosomes. The results are presentedin FIG. 2.

FIG. 2 is intended to show inactivation of certain grain hemitoxins bySPDP conjugation. It indicates that even 1:1 ratios of SPDP to hemitoxinresult in significant inactivation which is complete at a 20:1 ratio. Acommonly used 2-3 fold ratio would result in >95% inactivation.Applicants' study was expanded to include hemitoxins from corn andpokeweed. Reactions were carried out in phosphate buffers at neutral andacidic pH's in anticipation that under acidic conditions differences inpKa of lysine amino groups or conformational changes in some of theproteins might protect enzymic activity. However, in all conditions andwith all 4 hemitoxin proteins, significant inactivation occurred and asquantitative activity measurements of hemitoxins were rather imprecise;hence applicants were unable to conclude that residual activity was notfrom unreacted hemitoxin. Moreover, these particular experimentsindicated SPDP would be unsuitable as a coupling reagent for preparingmany GnRH-A-T conjugates.

4. Conjugation of [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide to toxins usingCarbodiimide. Applicants examined the ability of the water solublecoupling reagent, carbodiimide linkages in this class of compounds.Although carbodiimide has been used successfully for couplingpolypeptide hormones to proteins, applicants are unaware of any studiesreporting its use in preparing toxin-protein conjugates. However, itsuse turned out to be attractive since it couples through carboxyl groupson the hemitoxin rather than amino groups. It should also be noted thatapplicants' synthetic GnRH analogs are blocked at the carboxyl and aminotermini, thus leaving, for example, D-lys⁶ amine as the only reactivemoiety. Use of large molar ratios of GnRH favors reaction of thehemitoxin to the analog rather than to itself.

FIG. 3 shows the successful results of this approach. It represents aSDS-PAGE analysis of carbodiimide conjugated hemitoxins. In order tocarry out these experiments, a 30:1 molar ratio of [D-Lys⁶, des-Gly¹⁰]-GnRH-ethylamide to hemitoxin was reacted with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in water at 23° C.for 30 minutes and the reaction mixture passed through a Bio-Gel P6column to desalt the product. Protein containing fractions were assayedfor residual activity (see text) and the reaction products examined bySDS polyacrylamide gel electrophoresis. Lanes 1, and 6 are standards;lane 2, barley; lane 3, barley-GnRH; Lane 4, pokeweed; lane 5,pokeweed-GnRH; lane 7, rye-GnRH; lane 8, rye; lane 9, gelonin-GnRH; lane10, gelonin. Conjugation in each case resulted in a 32 kDa product whichwas distinct from the 30 kDa hemitoxin alone, and which (by enzymeassay) retained 10% of the original activity. Hemitoxins from barley,rye, wheat and the unrelated pokeweed and gelonin hemitoxins have eachbeen successfully conjugated in this fashion and all retain about 10% oforiginal toxicity in ascites ribosomal assay. Biologic studies withthese conjugates were then completed in the manner hereinafterdescribed.

5. Conjugation of [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide to toxins using2-iminothiolane. Although 2-iminothiolane, like SPDP, reacts with freeamino groups on proteins, it does not affect the activity of gelonin orPAP. Applicants have hypothesized that perhaps the reason2-iminothiolane differs from SPDP in this regard is that it reacts witha different amino group on the protein or that it places a positivecharge on the active amino group and thereby preserves enzymaticactivity. In any case, applicants reacted 2-iminothiolane with barleyhemitoxin at several reagent: protein ratios, separated the protein fromunreacted 2-iminothiolane by gel exclusion chromatography on SephadexG-25 and quantitated the amount of sulfhydryl groups introduced onto thehemitoxin by sulfhydryl exchange with the reactive, chromogenicdisulfide. 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). The derivatizedbarley hemitoxin preparations were assayed for their ability to inhibitprotein synthesis in ascites cell-free extracts and were found to haveretained full activity.

FIG. 4 depicts inhibition of protein synthesis by2-iminothiolane-conjugated barley hemitoxin. Barley hemitoxin wasincubated at 0° C. for 90 minutes with 0 (o), 8-fold (x) or 24-fold (o)molar excess of 2-iminothiolane. The derivatized hemitoxins were thenassayed for their ability to inhibit protein synthesis in ascitescell-free extracts. Proteins contained 0 (o), 0.76 (x) and 1.44 (o)moles of 2-iminothiolane bound per mole of hemitoxin.

Conjugation between the barley hemitoxin and [D-Lys⁶, des-Gly¹⁰]-GnRH-ethylamide was carried out by disulfide exchange. A sulfhydrylgroup was introduced into [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide byreacting the hormone with a 16- fold molar excess of 2-iminothiolane at0° C. for 2 hours. Derivatized [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide wasseparated from unreacted 2-iminothiolane by chromatography on a Bio-GelP-2 column equilibrated with 30% acetic acid. Acetic acid was removedfrom the isolated hormone by rotary evaporation followed bylyophilization. A reactive disulfide was prepared from barley hemitoxinas described above by incubating the hemitoxin with a 24-fold molarexcess of 2-iminothiolane, isolating the protein and reacting it withDTNB to prepare the disulfide, and separating the hemitoxin fromunreacted DTNB by column chromatography on Sephadex G-25. A 12-foldmolar excess of derivatized [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide wasadded to hemitoxin disulfide and disulfide exchange permitted to occurovernight at 4° C. Hemitoxin was separated from unconjugated GnRH bySephadex G-25 column chromatography.

The reaction products were analyzed by SDS-polyacrylamide gelelectrophoresis under non-reducing conditions. Analysis showed that thecoupling reaction had converted approximately 50% of the 29 kDa barleyhemitoxin (track 5) into a 31 kDa product (tracks 1-4) corresponding toa 1:1 hemitoxin-[D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide conjugate. Thefaint band of unreacted [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide that can beseen in track 1 migrating ahead of the 14 kDa marker disappearedfollowing acetone precipitation of the hemitoxin (track 2) or gelexclusion chromatography on Sephadex G-25 (tracks 3 & 4). The mixture ofconjugate and unreacted hemitoxin was not purified further but wasassayed directly for pituitary cell binding and killing.

FIG. 4A depicts SDS-PAGE analysis of barley hemitoxin after conjugationto [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide using 2-iminothiolane. Reactionproducts were analyzed before (tracks 1 & 2) and after tracks 3 & 4)Sephadex G-25 chromatography, and before (tracks 1 & 3) and after(tracks 2 & 4) concentrating by acetone precipitation. Track 5 containedunreacted hemitoxin.

6. Conjugate Binding Studies. In order to assess whether [D-Lys⁶,des-Gly¹⁰ ]-GnRH-ethylamide toxin conjugates retain their ability tobind to receptors, the following assay was devised. Variousconcentrations of each conjugate were evaluated for their ability todisplace 50,000 cpm ¹²⁵ I-D Ala⁶ -GnRH-ethylamide from bovine pituitarymembranes. After incubation for 4 hours in standard conditions at 4° C.,membranes were pelleted, counted in a gamma counter to determine thebound labelled ligand, and the ability of each conjugate to displace 50%of the label (IC₅₀ for unlabelled [D-Lys⁶, des-Gly¹⁰ ]-GnRH-ethylamide.FIG. 5 indicates the results of binding curves obtained in theseexperiments. Also shown are the calculated number of molecules requiredto displace 1 molecule of unconjugated [D-Lys⁶, des-Gly¹⁰]-GnRH-ethylamide. For example, FIG. 5 shows competitive binding oftoxin conjugates to bovine pituitary membranes. The abbreviations are:2IT, 2-iminothiolane; PAP, Pokeweed Antiviral Protein; SPDP,N-succinimidyl 3-(2-pyridyldithio) propionate; CI, Carbodiimide; EACA,Epsilon-amino caproic acid linker. Grain names refer to the purifiedhemitoxin source.

The data in FIG. 5 was critical in determining applicants' next steps.Several conclusions were reached. First, SPDP severely limits toxinactivity (see FIG. 2). It also produces conjugates with greatly reducedbinding activity (compare PAP-SPDP with Barley carbodiimide). On theother hand, use of carbodiimide produced conjugates with 3-40 foldimproved binding compared to SPDP. However, there were differences amongthe hemitoxins used. For example, the wheat, rye and gelonincarbodiimide conjugates all showed greater binding than did the barleycarbodiimide conjugate. However, the barley carbodiimide conjugateretained greater toxicity than the other grain hemitoxin conjugates inthe cell free protein synthesis assay (data not shown). In this case,use of a spacer arm actually decreased binding affinity. Finally, the2-iminothiolane conjugate made with barley hemitoxin as described aboveretained both 100% toxicity in the cell free system (see generally FIG.4) and was as active as the best of the carbodiimide conjugates inbinding. Applicants noted a 4.5 fold reduction in binding compared tothe unconjugated [D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide. This was quiteacceptable since native GnRH has also only about 1/30 the bindingactivity as this analogue (data not shown). Thus, after this exploratorywork was completed, applicants carried out most further work with eitherthe PAP-SPDP-[D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide or the barley2-imminothiolane [D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide conjugate.

In Vitro Experiments.

The effect of these compounds on ovine pituitary cells in suspensionculture was measured. A pituitary was removed from a ewe, sliced thinly,and dissociated with a mix of collagenase, hyaluronidase, and DNAase.The cells were washed several times and resuspended in culture mediumcontaining 30% ram's serum. Cells were cultured in a 37° shaking waterbath in 50 ml flasks under 95% O₂ /5% CO₂. In a typical experiment,cells were divided into four groups after dissociation and culturedovernight (20 hr) with 1) culture medium only, 2) 10⁻⁸ M GnRH, 3) 3×10⁻⁹M Toxin-[D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide (molarity expressed in termsof GnRH receptor binding activity) and 4) Toxin at the sameconcentration as Toxin-[D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide. Afterpretreatment, the cells were washed 6 times, counted, and small aliquotsremoved for testing. The remainder were cultured in plain medium for 24hours. To test the cells, aliquots of 500,000 cells were washed andresuspended in challenge medium containing 10⁻⁷ M GnRH for 2 hours at37° C. 3 ml of cold Gel-PBS was added to each tube, cells werecentrifuged, and the media was measured for LH content. The fourpretreatment groups were evaluated for their ability to synthesize andsecrete LH immediately after treatment and after the 24 hour recoveryperiod. The results of one experiment are shown in Table III.

                  TABLE III                                                       ______________________________________                                        LH Synthesis and Release by Ovine Pituitary Cells                             (ng per 5 × 10.sup.6 cells)                                             TREATMENT.sup.1 SYNTHESIS.sup.2                                               ______________________________________                                        CONTROL         526.3                                                         10.sup.-8 M GnRH                                                                              545.5                                                         PAP             137                                                           PAP-D-Lys.sup.6 0                                                             ______________________________________                                         .sup.1 Cells were incubated with the various treatments for 16 hours.         .sup.2 Synthesis of LH was measured during a 24 hour period of culture        after the agents were removed from the cells.                            

These data, although obtained with the least promising of ourconjugates, reveal a large and specific effect of PAP-SPDP-[D-Lys⁶,desGly¹⁰ ]-GnRH-ethylamide (ethylamide is abbreviated as "EA" in TableIII) on the gonadotropes ability to synthesize and secrete LH. It is notpossible to determine whether the gonadotropes were specifically killedas they comprise <10% of the total number of pituitary cells, but thedata strongly suggest the conjugate disrupted their normal function.

Applicants then tested the more promising Barley-2IT-[D-Lys⁶, desGly¹⁰]-GnRH-ethylamide conjugate in similar assay systems. Table IV shows theresults of a similar experiment. Ovine pituitary cells were again placedin culture with various agents and the total LH in the cells and mediadetermined after a 24 hour exposure, wash, and further 24 hour culturein standard media.

                  TABLE IV                                                        ______________________________________                                        Total Culture LH after Exposure to GnRH                                       and Toxin Conjugates with or without Lysosomal Agents                                              Total LH (Ng/10.sup.5                                    Incubation Condition cells in Culture)                                        ______________________________________                                        Control              1.90                                                     D-Lys.sup.6 GnRH--EA 1.62                                                     Barley Toxin         1.49                                                     Barley Toxin-2IT-D-Lys.sup.6 GnRH--EA                                                              .91                                                      Barley Toxin-2IT-D-Lys.sup.6 GnRH--EA +                                                            1.83                                                     Monensin                                                                      Barley Toxin-2IT-D-Lys.sup.6 GnRH--EA +                                                            .62                                                      Chloroquine                                                                   Barley Toxin-2IT-D-Lys.sup.6 GnRH--EA +                                                            1.33                                                     NH.sub.4 Cl                                                                   Barley Toxin-2IT-D-Lys.sup.6 GnRH--EA +                                                            1.13                                                     Killed Adenovirus                                                             ______________________________________                                    

These results indicate a specific killing effect of the toxin conjugateafter only 24 hours of exposure. The lysosomally active agents do notpotentiate this effect with the exception of chloroguine. When suchexperiments are combined with secondary challenge by GnRH, it appearsthat few gonadotropes are able to synthesize new LH after exposure tothe barley toxin conjugate (data not shown).

7. In vivo Experiments. Several experiments were done to determine theeffects of the pokeweed toxin (PAP) -SPDP-[D-Lys⁶ , desGly¹⁰]-GnRH-ethylamide conjugate in adult Sprague Dawley rats. Groups of 5-7rats were treated with 20 ng of analogue; 20 ng conjugate (receptorbinding assay equivalents), saline, or a conjugate made from a proteinof similar molecular weight to the pokeweed toxin (carbonic anhydrase orovalbumin). The most effective time course was found to be weeklyinjections for 4 weeks. The effect of such treatment was monitored inseveral ways. The ability of the animals to respond to a GnRH analoguechallenge by measuring LH and/or serum testosterone levels 30-90 minutesafter injection was followed. No difference was found among the groups.This result might be expected, since inducible LH release in intactanimals is quite small secondary to chronic feedback suppression by thetesticular androgens. Secondly, applicants followed gonad weights andfound the testes in the PAP-[D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide group tobe decreased by 50%, although the control conjugates had similareffects. The PAP-[D-Lys⁶, desGly¹⁰ ]-GnRH and carbonic anhydraseconjugate groups were found to be infertile in breeding tests,indicating a potential effect of this enzyme on testis tissue.Interestingly, light microscopy of these animals revealed no changes inthe pituitaries, but interstitial (Leydig) cell depletion in thePAP-SPDP-[D-Lys⁶, desGly¹⁰ ]-GnRH-ethylamide treated group, indicating apossible specific cellular effect on rat testicular function. This wasnot surprising since there are GnRH receptors on Leydig cells in the rattestis.

Applicants also tested the PAP-carbodiimide-[D-Lys⁶, desGly¹⁰]-GnRH-ethylamide conjugate in ovariectomized female rats. In contrastto the SPDP conjugate, and in this system where gonadal feedback is nota problem, this drug appears capable of producing a 15 fold decrease inthe serum LH response to GnRH analogue challenge (FIG. 1A or 1B), againindicating the importance of applicant's studies on various linkingtechniques.

FIG. 1B indicates the results of a challenge by one of applicants'compounds to ovariectomized rats. Serum concentrations of LH inovariectomized rats treated with saline (hatched bars) or pokeweedanti-viral protein conjugated to a GnRH super-agonist (solid bars) aredepicted. The open space above the bars indicates the amount of LHreleased in response to a GnRH challenge. The challenges wereadministered on the first day of treatment and again 4 weeks later.Compared to control there was greater than a 90% reduction in LH releaseafter GnRH challenge at 4 weeks of treatment.

Based on the above data (with regards to LH synthesis inhibition)applicants then carried out experiments in intact male and female rats.Animals received 4 injections at 3 day intervals of PAP-CI-[D-Lys⁶,desGly¹⁰ ]-GnRH-ethylamide or of the GnRH analogue or toxin alone orsaline. Conjugate treated male animals (but not control) showed a 50%reduction in fertility (i.e., 50% of females exposed to these maleanimals became pregnant, compared to 100% of controls). Histologicexamination of the testes of experimental animals revealed residualspermatogenesis in about 10% of tubules. In conjugate treated femaleanimals, fertility was abrogated for more than 4 months (time sufficientfor about 30 reproductive cycles in normal animals) following treatment.There were no side effects noted from these injections.

To further understand the effect of hemitoxins and conjugates onnon-target tissues, applicants initiated studies on the tissuedistribution of ¹²⁵ I-toxin-conjugates and have demonstrated importantdifferences among the toxins in (for example) concentration in thekidneys, indicating the importance of testing the various proteins toavoid potential non-target tissue toxicity. For example, applicants havefound that the tissue/serum ratio of unconjugated PAP 2 hours afterinjection for various organs ranges from 0.03 in brain to 85.5 inkidney. In contrast, unconjugated barley hemitoxin is 8-fold lessconcentrated in kidney (see Table IV). Conjugation with the GnRHanalogue alters these ratios considerably.

                  TABLE V                                                         ______________________________________                                        Tissue Distribution of Hemitoxins                                             and Hemitoxin Conjugates                                                      Tissue      PAP     PAP-SPDP-D-Lys.sup.6 GnRH                                 ______________________________________                                        Pituitary   .20     .11                                                       Brain       .03     .01                                                       Adrenal     .48     .02                                                       Kidney      85.5    12.6                                                      Liver       2.48    1.07                                                      Spleen      2.29    .73                                                       Testis      .03     .02                                                       ______________________________________                                        Tissue/serum Ratio of Labeled Protein                                         GnRH        Barley  Barley-CI-D-Lys.sup.6 GnRH                                ______________________________________                                        Pituitary   1.08    1.06                                                      Brain       .04     .04                                                       Adrenal     .70     1.5                                                       Kidney      10.5    4.0                                                       Liver       .43     3.52                                                      Spleen      .4      5.07                                                      Testis      .10     .10                                                       ______________________________________                                    

Thus these experiments produced a group of compounds capable ofsterilizing (temporarily or permanently) animals by destroying thegonadotrophs of an animal's anterior pituitary gland. These compoundscan be administered in the form of pharmaceutically acceptable, andotherwise nontoxic salts. It should also be noted that these compoundscan be administered individually, or in combination with each other, toanimals intravenously, subcutaneously, intramuscularly or orally toachieve fertility inhibition and/or control. Preferably administrationwill be intravenous or intramuscular in a suitable carrier such as, forexample, in isotonic saline phosphate buffer solutions or the like. Theyalso can be used in applications calling for reversible suppression ofgonadal activity, such as for the management of precocious puberty orduring radiation or chemotherapy. Effective dosages will vary with theform of administration and the particular species of mammal beingtreated. An example of one typical dosage form is a physiological salinesolution containing the peptide which solution is administered toprovide a dose in the range of about 0.1 to 10 mg/kg of body weight.

Although the invention has been described with regard to its preferredembodiments, it will be apparent to those skilled in this art, uponreading the above, detailed description and examples, that variousmodifications and extensions can be made thereto without departing fromthe spirit of the present invention and that the scope of said inventionshall be limited only by the scope of the appended claims.

Thus having disclosed this invention, what is claimed is:
 1. A methodfor functionally inactivating gonadotrophs in the pituitary gland of ananimal, comprising administering to said animal an effective amount of ahormone/toxin conjugate comprising a peptide hormone conjugated to atoxin group, wherein said conjugate is capable of selectively bindingwith receptors on said gonadotrophs to render said gonadotrophsessentially incapable of secreting gonadotropins, wherein said animal isnot weakened or killed by said method.
 2. A method of claim 1, whereinsaid method is effective to temporarily sterilize said animal.
 3. Amethod of claim 1, wherein said method effects permanent sterilizationof said animal.
 4. A method of claim 1, wherein said method is effectivefor treating a sex hormone related disease in said animal.
 5. A methodof claim 1, wherein said peptide hormone is GnRH or an analog thereof.6. A method of claim 1, wherein said peptide hormone has the generalformula

    pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Z,

wherein X is an amino acid selected from the group consisting of lysine,D-lysine, ornithine, D-ornithine, glutamic acid, D-glutamic acid,aspartic acid, D-aspartic acid, cysteine, D-cysteine, tyrosine andD-tyrosine; and Z is a substituent selected from the group consisting ofGly-NH₂, ethylamide, and AzA-Gly-NH₂.
 7. A method of claim 1, whereinsaid peptide hormone comprises [D-Lys⁶ -des-Gly¹⁰ ]-GnRH-ethylamide. 8.A method of claim 1, wherein said toxin group is a modified toxincomprising a toxic domain and a translocation domain but lacking afunctional toxin cell binding domain.
 9. A method of claim 1, whereinsaid toxin group is a toxin selected from the group consisting of achemical toxin, a single chain toxin, and a modified toxin having anintrinsic toxic group and intrinsic translocation domain but lacking afunctional intrinsic toxin cell binding domain.
 10. A method of claim 9,wherein said modified toxin is selected from the group consisting ofmodified ricin toxins, modified modeccin toxins, modified abrin toxins,modified diphtheria toxins, modified Pseudomonas exotoxins and modifiedshiga toxins.
 11. A method of claim 9, wherein said single chain toxinis selected from the group consisting of pokeweed antiviral protein,α-amanitin, gelonin ribosome inhibiting protein ("RIP"), barley RIP,wheat RIP, corn RIP, rye RIP, flax RIP, and modified forms thereof. 12.A method of claim 9, wherein said chemical toxin is selected from thegroup consisting of melphalan, methotrexate, nitrogen mustard,doxorubicin and daunomycin.
 13. A method of claim 1, wherein said toxingroup is selected from the group consisting of modified diphtheriatoxins and modified Pseudomonas exotoxins, wherein said toxin groupcomprises a toxic domain and a translocation domain but lacks afunctional toxin cell binding domain.
 14. A method of claim 1, whereinsaid peptide hormone is conjugated to said toxin group using a linkingagent capable of forming a stable conjugate that does not substantiallydegrade prior to binding with said gonadotrophs but which is capable ofreleasing a sufficient amount of said toxin group from said conjugate inthe cytosol of said gonadotrophs to substantially preclude secretion ofgonadotropins by said gonadotrophs.
 15. A method of claim 14, whereinsaid linking agent is selected from the group consisting of2-iminothiolane, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),4-succinimidyloxycarbonyl-α-(2-pyridyldithio)toluene (SMPT),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-diazobenzidineand glutaraldehyde.
 16. A method of claim 14, wherein said linking agentis selected from the group consisting of SMPB, 2-iminothiolane, and EDC.17. A method of claim 1, further comprising challenging said animal withGnRH at least about four weeks after said step of administering saidconjugate to said animal, said challenging not inducing substantialsecretion of luteinizing hormone by said animal.
 18. A method of claim1, wherein said method of administering said conjugate is repeated atleast once over a time period such that administration of said conjugatedoes not elicit a substantial production of antibodies against saidconjugate.
 19. A method of claim 1, wherein said conjugate has thegeneral formula ##STR4## wherein Y comprises SMPB and wherein T is atoxin group selected from the group consisting of modified diphtheriatoxins and modified Pseudomonas exotoxins, wherein said toxin groupcomprises a toxic domain and a translocation domain but lacks afunctional toxin cell binding domain.
 20. The method of claim 19,wherein said sex hormone related disease is selected from the groupconsisting of breast cancer, prostate cancer, pancreatic cancer, andendometriosis.
 21. The method of claim 1, wherein said conjugate caninteract with GnRH receptors and gain entry into cells presenting saidreceptors.
 22. A method for chemically attacking cells whose membranescontain receptors for GnRH, comprising administering to an animal aneffective amount of a hormone/toxin conjugate comprising a peptidehormone that is capable of binding to a GnRH receptor, said peptidehormone conjugated to a toxin group, wherein said conjugate is capableof selectively binding to said GnRH receptors on said cells.
 23. Themethod of claim 22, wherein said peptide hormone is GnRH or an analogthereof.
 24. The method of claim 22, wherein said peptide hormone hasthe general formula

    pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Z,

wherein X is an amino acid selected from the group consisting of lysine,D-lysine, ornithine, D-ornithine, glutamic acid, D-glutamic acid,aspartic acid, D-aspartic acid, cysteine, D-cysteine, tyrosine andD-tyrosine; and Z is a substituent selected from the group consisting ofGly-NH₂, ethylamide, and AzA-Gly-NH₂.
 25. The method of claim 22,wherein said toxin group is a modified toxin comprising a toxic domainand a translocation domain but lacking a functional toxin cell bindingdomain.
 26. The method of claim 22, wherein said toxin group is a toxinselected from the group consisting of a chemical toxin, a single chaintoxin, and a modified toxin having an intrinsic toxic group andintrinsic translocation domain but lacking a functional intrinsic toxincell binding domain.
 27. The method of claim 22, wherein said conjugatehas the general formula ##STR5## wherein Y comprises SMPB and wherein Tis a toxin group selected from the group consisting of modifieddiphtheria toxins and modified Pseudomonas exotoxins, wherein said toxingroup comprises a toxic domain and a translocation domain but lacks afunctional toxin cell binding domain.